Restriction endonucleases are a class of enzymes which occur naturally in prokaryotic and eukaryotic organisms. When they are purified away from other contaminating cellular components, restriction endonucleases are used in the laboratory to cleave DNA molecules into precise fragments. Thus, restriction endonucleases have proved to be indispensable tools in modern genetic research.
Restriction endonucleases cleave DNA by first recognizing and binding to particular sequences of nucleotides (the "recognition sequence") along the DNA molecule. The enzyme cleaves both strands of the DNA molecule within, or to one side of, this recognition sequence. The cleavage site can be blunt ended, both of the DNA strands are cleaved at the same nucleotide, or cleaved asymmetrically to yield a 3' or 5' overhang. Restriction endonucleases isolated from different sources most often have an affinity for different recognition sequences. About 100 kinds of different endonucleases have so far been isolated, many from microorganisms. Each restriction endonuclease is identified by its recognition sequence and by the cleavage pattern it exhibits when a target DNA, i.e. pBR 322, is digested by the particular enzyme.
A number of restriction endonucleases, called restriction endonuclease isoschizomers, have been isolated from unrelated microorganisms. Isoschizomers are enzymes which recognize the same recognition sequence. In some cases, though recognizing the same recognition sequence, the isoschizomers may cleave at different phosphodiester bonds within or to one side of the recognition sequence.
Modification methylases, complementary to their corresponding restriction endonucleases, recognize and bind to the same recognition sequence. Modification methylases chemically modify certain nucleotides within the recognition sequence by the addition of a methyl group, rendering the sequence resistant to cleavage by the complementary restriction endonuclease. Thus, in nature, methylases serve a protective function, i.e., to protect the DNA of an organism which produces the complementary restriction enzyme.
Restriction enzymes and modification methylases can be purified from the host organism by growing large amounts of cells, lysing the cell walls, and purifying the specific enzyme away from the other host proteins by extensive column chromatography. However, the amount of restriction enzyme relative to that of the other host proteins is usually quite small. Thus, the purification of large quantities of restriction enzymes or methylases by this method is labor intensive, inefficient, and uneconomical. By cloning the genes encoding for the desired restriction and modification enzymes and over expressing them in a well studied organism, generally Escherichia coli (E. coli), the amount of these enzymes, relative to that of the host proteins, may be increased substantially.